Without limiting the scope of the invention, its background is described in connection with the design and delivery of shRNA-based therapies targeting the expression of the PDX-1 oncogene as treatments for pancreatic neuroendocrine tumors.
Islet neoplasia refers collectively to disorders emanating from pancreatic islet cells, such as pancreatic neuroendocrine tumors (NET), insulinomas, and carcinoid. Islet cell tumors have a 5-year survival rate between 35-50% [1,2]. Islet neoplasia affects relatively young patients (age at diagnosis 50 years) [3,4]. Patients with insulinoma in particular suffer horribly from uncontrollable hypoglycemia, for which there is no effective treatment. Hypoglycemia results from hyperinsulinemia and manifests with symptoms of neuroglycopenia, such as headache, dizziness, lethargy, diplopia, seizures and sympathetic activation [3]. Surgery remains the most effective treatment modality, and there are no effective adjuvant therapies. When synchronous resection of the primary tumor and metastases is possible, recurrence rates remain high. Moreover, survival has not improved with systemic therapy [5,6]. Thus, a need exists for novel and effective therapeutic approaches to treat NET.
Pancreatic duodenal homeobox-1 (PDX-1) is an embryonic transcription factor of pancreatic organogenesis that is over expressed in most NET's and all islet cell insulinomas. The transcription factor PDX-1 gene is directly responsible for regulating insulin secretion in benign and malignant insulinomas [7-10]. It is also found in a variety of cancers where it appears to be associated with proliferation, differentiation, and migration, with a primary role in the development of NET and pancreatic embryogenesis [7-14]. In the adult pancreas, PDX-1 is expressed by 90% of β cells, where it has been shown to regulate expression of insulin, glucokinase, and glucose transporter type 2 (GLUT2) [15-19]. These genes have a critical role in maintaining glucose homeostasis in both acute and chronic hyperglycemia. In acute hyperglycemia, PDX-1 migrates from the cytoplasm to the nucleus of β-cells and activates the insulin gene, the first step in increased insulin production and secretion [20, 21].
PDX-1 was shown to be overexpressed in a number of cancers by the present inventors and others [22-25]. In particular, the present inventors found that PDX-1 was overexpressed in more than 80 pancreatic cancers and islet cell neoplasias [26]. One hundred percent of insulinomas also have PDX-1 overexpression. The inventors have recently demonstrated that PDX-1 regulates proliferation and invasion of pancreatic cancer and insulinoma cell lines [27]. It will be appreciated by those skilled in the art that if the compositions and methods to modulate the overexpression of PDX-1 in cancers are provided, such compositions and methods would have a positive value and represent a substantial contribution to the art. Likewise, it will be appreciated by those versed in the drug delivery art that if a delivery system is provided to bypass non-target organs and target the delivery of the therapeutic agent to the target organs over expressing PDX-1, such a delivery system would be clinically useful in the practice of medicine. A strategy for treating pancreatic adenocarcinoma by targeting PDX-1 is provided in U.S. Pat. No. 6,716,824 issued to Brunicardi (2004) that relates to a recombinant nucleic acid for an RIP-tk (rat insulin promoter-thymidine kinase) construct that selectively targets insulin secreting cells, such as β-cells, PDX-1 positive human pancreatic ductal carcinomas, and other cells containing certain transcription factors. The Brunicardi invention is useful in the treatment of pancreatic cancers, such as β-cell insulinomas.
Preclinical studies confirm that RNA interference techniques (RNAi) can be used to silence cancer-related targets [28-38]. In vivo studies have also shown favorable outcomes by RNAi targeting of components critical for tumor cell growth [29, 39-42], metastasis [43-45], angiogenesis [46, 47], and chemoresistance [48-50]. Applications of the RNAi technology initially employed two types of molecules; the chemically synthesized double-stranded small interfering RNA (siRNA) or vector based short hairpin RNA (shRNA). Although siRNA and shRNA can achieve similar knockdown functions, siRNA and shRNA are intrinsically different molecules. shRNAs, which share the same biogenesis pathways as the naturally occurring microRNAs (miRNAs), are processed and transported to the cytoplasm where they load onto the RNA interference silencing complexes (RISC) [51]. The primary transcripts of endogenous miRNAs are synthesized from genomic DNA in the nucleus as long RNA strands with hairpin structures, which are processed to mature 21-23 base pair double stranded miRNA by a series of endogenous RNase III enzymes. At least two RNase III enzyme complexes are involved in the maturational process; first, the Drosha/DGCR8 complex produces the pre-miRNA hairpin structure which, following nuclear export, is incorporated into the RLC where the second enzyme, Dicer, excises the loop producing the mature miRNA [52], the effector molecule that is loaded onto RISC for RNAi [53].
The Argonaute (Ago) family of proteins commonly associates with the cytoplasmic RISC. These proteins are involved in the loading of siRNA or miRNA, and are also implicated in both transcriptional (targeting heterochromatin) and post-transcriptional gene silencing. The guide strand of siRNA or miRNA loaded onto Ago-complexed RISCs seeks out target mRNAs with sequence complementarity. Endonucleolytically active Ago-2 cleaves mRNA to initiate mRNA degradation [54, 55]. Alternatively, partial complementary binding to the 3′ UTR of the targeted mRNA achieves translational repression through sequestration in processing bodies (P-bodies) [56]. Deadenylation leading to destabilization of the target mRNA has been observed in P-bodies [57, 58]. There is now compelling evidence in numerous animal models that RNAi-mediated gene knockdown is potent, specific, and well tolerated. These findings provide the scientific rationale for the translation of RNAi therapeutics into the clinic. At least 10 RNAi-based drugs are now in early phase clinical trials [59], four of which are cancer related [60-63]. Additional siRNAs have also demonstrated efficacy in animal models [60,61,64] and safety in non-human primates [65].
U.S. Patent Application No. 2009/0163431 (Kazhdan et al., 2009) discloses methods and compositions for inhibiting PDX-1. An anti-PDX-1 agent included in inventive methods and compositions that includes an antibody, an aptamer, an antisense oligonucleotide, a ribozyme and/or an inhibitory compound. Methods of inhibiting PDX-1 expression in a tumor cell are provided by the present invention which include contacting a tumor cell with an effective amount of an anti-PDX-1 agent highly or completely complementary to a specified region of an RNA molecule encoding PDX-1. Such an agent specifically hybridizes with the RNA molecule encoding PDX-1 and inhibits the expression of a PDX-1 gene in the tumor cell. Compositions including anti-PDX-1 siRNA and/or shRNA are described. Recombinant expression constructs encoding anti-PDX-1 siRNA or shRNA according to the present invention are described.